The establishment of polarity and provision of spatial cues in the embryo are prerequisites for the development of a complex multicellular organism. These requirements are essentially problems of localization, or how to position morphogens or spatial cues non randomly in a cell or group of cells. The long term goal of this application is to understand one prominent example of the localization phenomenon, that of the bicoid morphogen. The bicoid gene is largely responsible for anterior body patterning in the Drosophila embryo. Appropriate spatial deployment of the bicoid protein morphogen in the anterior of the embryo relies on the prelocalization of bicoid mRNA to the anterior pole of the egg during oogenesis. Similar mRNA prelocalizations are involved in deployment of other patterning molecules; additional prominent examples come from both Drosophila and Xenopus. The specific aims of this application fall into two related areas. First, we will continue our identification and characterization of the cis-acting elements within the bicoid mRNA that direct its localization. One previously identified element, BLEI, will be scrutinized in detail. A collection of mutated forms of BLEI will be constructed by in vitro DNA manipulations. These will be tested by a rapid in vitro assay for binding to an ovarian protein, pi 15, that binds specifically to wild-type BLEI and is a strong candidate to act in bicoid mRNA localization. Some of the same BLEI mutants will be tested for their abilities to direct localization in vivo using transgenic fly strains. From the behavior of the BLEI mutants we hope to learn how BLEI acts in localization. BLEI does not direct all stages of localization, and so we will attempt to identify and define the missing localization elements, monitoring the localization properties of mutants lacking parts of the bicoid mRNA other than BLEI. The second direction of the work is to isolate and characterize trans-acting factors involved in bicoid mRNA localization. Efforts will initially be directed towards two protein factors. One is the previously mentioned ovarian BLEI-binding protein. We will purify the protein and clone the gene that encodes it. Molecular and genetic characterization of the gene will be undertaken to explore its role in localization, with the eventual aim of asking what molecules it contacts in addition to bicoid mRNA. The other protein factor is the product of the exuperantia gene, which we have cloned. Because our results suggest that exu protein makes interactions with other proteins, we will use a yeast screening system to identify and isolate those proteins. Collectively, this work will provide a strong foundation for understanding how bicoid mRNA is localized.